Description
Human NADH-cytochrome b5 reductase 3 (CYB5R3) ELISA Kit
The Human NADH-Cytochrome B5 Reductase 3 (CYB5R3) ELISA Kit is a powerful tool designed for the quantitative detection of CYB5R3 levels in human samples such as serum, plasma, and cell culture supernatants. This kit boasts exceptional sensitivity and specificity, ensuring accurate and reproducible results for a wide range of research applications.CYB5R3 is a key enzyme involved in electron transport and lipid metabolism, playing a critical role in various physiological processes. Dysregulation of CYB5R3 has been linked to various diseases, including metabolic disorders, neurological conditions, and cancer.
As such, the measurement of CYB5R3 levels can provide valuable insights into disease pathogenesis and aid in the development of potential therapeutic interventions.With its reliable performance and versatility, the Human NADH-Cytochrome B5 Reductase 3 ELISA Kit is a valuable tool for researchers studying the role of CYB5R3 in health and disease. Stay ahead of the curve with this cutting-edge kit, delivering accurate and insightful results for your research needs.
| Product Name: | Human NADH-cytochrome b5 reductase 3 (CYB5R3) ELISA Kit |
| SKU: | HUEB0549 |
| Size: | 96T |
| Target: | Human NADH-cytochrome b5 reductase 3 (CYB5R3) |
| Synonyms: | Diaphorase-1, B5R, DIA1 |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Human |
| Detection Range: | 0.78-50ng/mL |
| Sensitivity: | 0.414ng/mL |
| Intra CV: | 5.9% | ||||||||||||||||||||
| Inter CV: | 8.5% | ||||||||||||||||||||
| Linearity: |
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| Recovery: |
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| Function: | Desaturation and elongation of fatty acids, cholesterol biosynthesis, drug metabolism, and, in erythrocyte, methemoglobin reduction. |
| Uniprot: | P00387 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant human NADH-cytochrome b5 reductase 3 |
| Sub Unit: | Component of a complex composed of cytochrome b5, NADH-cytochrome b5 reductase (CYB5R3) and MOSC2. |
| Research Area: | Cardiovascular |
| Subcellular Location: | Isoform 2 Cytoplasm Produces the soluble form found in erythrocytes. |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | CYB5R3: Desaturation and elongation of fatty acids, cholesterol biosynthesis, drug metabolism, and, in erythrocyte, methemoglobin reduction. Defects in CYB5R3 are the cause of methemoglobinemia CYB5R3-related (METHB-CYB5R3). A form of methemoglobinemia, a hematologic disease characterized by the presence of excessive amounts of methemoglobin in blood cells, resulting in decreased oxygen carrying capacity of the blood, cyanosis and hypoxia. There are two types of methemoglobinemia CYB5R3-related. In type 1, the defect affects the soluble form of the enzyme, is restricted to red blood cells, and causes well- tolerated methemoglobinemia. In type 2, the defect affects both the soluble and microsomal forms of the enzyme and is thus generalized, affecting red cells, leukocytes and all body tissues. Type 2 methemoglobinemia is associated with mental deficiency and other neurologic symptoms. Belongs to the flavoprotein pyridine nucleotide cytochrome reductase family. 3 isoforms of the human protein are produced by alternative promoter. |
| UniProt Protein Details: | Protein type:EC 1.6.2.2; Carbohydrate Metabolism - amino sugar and nucleotide sugar; Oxidoreductase; Mitochondrial Chromosomal Location of Human Ortholog: 22q13.2 Cellular Component: mitochondrial outer membrane; endoplasmic reticulum membrane; mitochondrion; membrane; endoplasmic reticulum; hemoglobin complex; cytoplasm; mitochondrial inner membrane; lipid particle Molecular Function:FAD binding; cytochrome-b5 reductase activity; ADP binding; NAD binding; AMP binding Biological Process: vitamin metabolic process; blood circulation; L-ascorbic acid metabolic process; cholesterol biosynthetic process; water-soluble vitamin metabolic process Disease: Methemoglobinemia Due To Deficiency Of Methemoglobin Reductase |
| NCBI Summary: | This gene encodes cytochrome b5 reductase, which includes a membrane-bound form in somatic cells (anchored in the endoplasmic reticulum, mitochondrial and other membranes) and a soluble form in erythrocytes. The membrane-bound form exists mainly on the cytoplasmic side of the endoplasmic reticulum and functions in desaturation and elongation of fatty acids, in cholesterol biosynthesis, and in drug metabolism. The erythrocyte form is located in a soluble fraction of circulating erythrocytes and is involved in methemoglobin reduction. The membrane-bound form has both membrane-binding and catalytic domains, while the soluble form has only the catalytic domain. Alternate splicing results in multiple transcript variants. Mutations in this gene cause methemoglobinemias. [provided by RefSeq, Jan 2010] |
| UniProt Code: | P00387 |
| NCBI GenInfo Identifier: | 127846 |
| NCBI Gene ID: | 1727 |
| NCBI Accession: | P00387.3 |
| UniProt Secondary Accession: | P00387,O75675, Q8TDL8, Q8WTS8, Q9UEN4, Q9UEN5, Q9UL55 Q9UL56, B1AHF2, B7Z7L3, |
| UniProt Related Accession: | P00387 |
| Molecular Weight: | 38,358 Da |
| NCBI Full Name: | NADH-cytochrome b5 reductase 3 |
| NCBI Synonym Full Names: | cytochrome b5 reductase 3 |
| NCBI Official Symbol: | CYB5R3 |
| NCBI Official Synonym Symbols: | B5R; DIA1 |
| NCBI Protein Information: | NADH-cytochrome b5 reductase 3; diaphorase-1; NADH-cytochrome b5 reductase 3 soluble form; NADH-cytochrome b5 reductase 3 membrane-bound form |
| UniProt Protein Name: | NADH-cytochrome b5 reductase 3 |
| UniProt Synonym Protein Names: | Diaphorase-1Cleaved into the following 2 chains:NADH-cytochrome b5 reductase 3 membrane-bound form; NADH-cytochrome b5 reductase 3 soluble form |
| Protein Family: | NADH-cytochrome b5 reductase |
| UniProt Gene Name: | CYB5R3 |
| UniProt Entry Name: | NB5R3_HUMAN |
| Component | Quantity (96 Assays) | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | -20°C |
| Lyophilized Standard | 2 | -20°C |
| Sample Diluent | 20ml | -20°C |
| Assay Diluent A | 10mL | -20°C |
| Assay Diluent B | 10mL | -20°C |
| Detection Reagent A | 120µL | -20°C |
| Detection Reagent B | 120µL | -20°C |
| Wash Buffer | 30mL | 4°C |
| Substrate | 10mL | 4°C |
| Stop Solution | 10mL | 4°C |
| Plate Sealer | 5 | - |
Other materials and equipment required:
- Microplate reader with 450 nm wavelength filter
- Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
- Incubator
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
| Step | |
| 1. | Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C. |
| 2. | Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform. |
| 3. | Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper. |
| 4. | Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C. |
| 5. | Repeat the wash process for five times as conducted in step 3. |
| 6. | Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction. |
| 7. | Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. |
| 8. | Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters. |
| 9. | After experiment, store all reagents according to the specified storage temperature respectively until their expiry. |
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
| Sample Type | Protocol |
| Serum | If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. |
| Plasma | Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit. |
| Urine & Cerebrospinal Fluid | Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid. |
| Cell culture supernatant | Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately. |
| Cell lysates | Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Tissue homogenates | The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C. |
| Tissue lysates | Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Breast Milk | Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles. |
